Small Animal MRI.

KEY WORDS: animal model; magnetic resonance imaging; thiamine deficiency; brain damage; AOD abstinence; AODE (alcohol and other drug effects) AIcoholism and other diseases affect the structure and function of many organs in the human body, including the brain. To understand the causes and to diagnose and track the progress of such diseases, scientists must understand the structural and functional changes in the affected organs. For many studies, particularly those involving the brain, researchers rely on noninvasive methods to visualize the organ under investigation. One technique commonly used to obtain images of a patient's brain or other area of the body is magnetic resonance imaging (MRI). This technique exposes the subject to radio waves in the presence of a strong magnetic field and then measures how the atoms that make up the body's tissues absorb the radio waves' energy. The manner of absorption varies, depending on the composition of the tissues (e.g., their water or fat content); with the help of computers, these variations can be converted into images. This article describes how MRI technology can be used not only on human patients but also in studies of laboratory animals that frequently serve as experimental models for human diseases. Also presented are findings of MRI studies that analyzed the effects of thiamine deficiency--the lack of a vitamin required for normal brain functioning--on the brain structure of rats. Preliminary findings indicate that similar studies can be used to assess the effects of long-term alcohol consumption and withdrawal on the brain. Although MRI has been used successfully in humans to diagnose diseases and study alterations in organ structures, its applications in elucidating the cause of a pathology or functional abnormality are more limited. To investigate the cause of a disease, researchers ideally would compare images of patients taken before and after they develop the disorder. Only rarely, however, do patients have images available for comparison that were taken prior to an illness. Alternatively, investigators can compare one group of subjects with a disease (e.g., alcoholics) with another group of people without the disease (e.g., nonalcoholics) to identify consistent differences between the two groups that might shed light on the cause of alcoholism. The subjects in such studies, however, will have different characteristics in addition to their alcohol intake (e.g., their medical and drinking histories and their genetic makeup). To make valid observations and to control for all possible variations among subjects, researchers must examine a large number of patients before attributing specific differences between the two study groups to alcohol use. Because of these limitations of human studies, researchers frequently rely on animal models, often using rats and other small animals. In general, animal studies provide more consistent information with fewer subjects than do human studies. The animals can be examined both before and after they develop a certain disease or undergo a particular treatment. Furthermore, animal studies can be rigidly controlled so that a particular change in organ structure or function can be directly attributed to the modification of a single experimental variable. Differences between animals can be minimized by using animals from one family (i.e., strain), a method that in humans would be feasible only with identical twins. And in contrast to studies on humans, studies using animals can be conducted across an animal's life span (about 2.5 years for rats), allowing the investigation of age-related effects. Conventional Techniques Histological techniques (i.e., studies that determine the structure of organs by examining thin tissue slices under a microscope) are commonly used to follow disease development in animals. Although these techniques allow researchers to view directly the tissue under investigation, they have several drawbacks, especially in studies of the brain. …

A nence; AODE (alcohol and other drug effects) lcoholism and other diseases affect the structure and function of many organs in the human body, includ ing the brain. To understand the causes and to diagnose and track the progress of such diseases, scientists must understand the struc tural and functional changes in the affected organs. For many studies, particularly those involving the brain, researchers rely on non invasive methods to visualize the organ under investigation. One technique com monly used to obtain images of a patient's brain or other area of the body is magnetic resonance imaging (MRI). This technique exposes the subject to radio waves in the presence of a strong magnetic field and then measures how the atoms that make up the body's tissues absorb the radio waves' en ergy. The manner of absorption varies, de pending on the composition of the tissues (e.g., their water or fat content); with the help of computers, these variations can be converted into images.
This article describes how MRI tech nology can be used not only on human patients but also in studies of laboratory ani mals that frequently serve as experimental models for human diseases. Also presented are findings of MRI studies that analyzed the effects of thiamine deficiency-the lack of a vitamin required for normal brain functioning-on the brain structure of rats. Preliminary findings indicate that similar studies can be used to assess the effects of longterm alcohol consumption and with drawal on the brain.
Although MRI has been used success fully in humans to diagnose diseases and study alterations in organ structures, its ap plications in elucidating the cause of a path ology or functional abnormality are more limited. To investigate the cause of a dis ease, researchers ideally would compare im ages of patients taken before and after they develop the disorder. Only rarely, however, do patients have images available for com parison that were taken prior to an illness. Alternatively, investigators can compare one group of subjects with a disease (e.g., alcoholics) with another group of people without the disease (e.g., nonalcoholics) to identify consistent differences between the two groups that might shed light on the cause of alcoholism. The subjects in such studies, however, will have different char acteristics in addition to their alcohol intake (e.g., their medical and drinking histories and their genetic makeup). To make valid observations and to control for all possible variations among subjects, researchers must examine a large number of patients before attributing specific differences between the two study groups to alcohol use.
Because of these limitations of human studies, researchers frequently rely on ani mal models, often using rats and other small animals. In general, animal studies provide more consistent information with fewer subjects than do human studies. The animals can be examined both before and after they develop a certain disease or undergo a parti cular treatment. Furthermore, animal studies can be rigidly controlled so that a particular change in organ structure or function can be directly attributed to the modification of a single experimental variable. Differ ences between animals can be minimized by using animals from one family (i.e., strain), a method that in humans would be feasible only with identical twins. And in contrast to studies on humans, studies using animals can be conducted across an animal's life span (about 2.5 years for rats), allowing the investigation of agerelated effects.

Conventional Techniques
Histological techniques (i.e., studies that determine the structure of organs by ex amining thin tissue slices under a micro scope) are commonly used to follow dis ease development in animals. Although these techniques allow researchers to view directly the tissue under investigation, they have several drawbacks, especially in stud ies of the brain.
First, the animals must be euthanized to remove small pieces of tissue for prepara tion before examination. Second, constraints of both time and expense generally restrict such studies to only one tissue and a few experimental parameters; consequently, the analyses could fail to detect the full effects of a disease or experimental procedure.
Third, because the animals are eutha nized to perform the measurement, each animal can be assessed only at a single timepoint during disease. To examine the entire disease process, researchers must study multiple animals at different stages of disease development and thus must esti mate the best timeframe for examining each animal. Because animals can vary significantly in the rate of disease progress, these estimates may miss their mark. For example, one rat may reach a particular stage of a disease 2 weeks earlier than an other rat, leading to wide variability in data.
To eliminate some of these problems and to take full advantage of the potential of ani mal studies, scientists have adapted nonin vasive techniques used in humans, such as MRI, for use in small animals (Henkelman et al. 1987;Fiel and Button 1990;Ballon et al. 1989;Yang et al. 1996). Small animal MRI (SAMRI) technology now has become so refined that it can produce very detailed images of an animal's brain or other organs.

Contributions of SAMRI
Like MRI in humans, SAMRI is a noninva sive technique. Its primary advantage over other procedures is that it is performed in living animals and therefore can be repeated several times in the same animal to track a disease's progress. Experiments that pre viously required hundreds of animals now may only use a dozen. Other advantages of this technique include: Comparable stages of disease progression can be pinpointed among several animals; tissue changes can be viewed as a dynamic process, exactly as they appear in a living animal; three dimensional relationships in tissue struc tures that are not evident in histological studies may be examined; researchers can examine more than one tissue simultane ously and thus detect and analyze con current and/or related changes in several tissues; and computerized image analysis allows researchers to measure quantitatively the tissues of interest.

How Is SAMRI Done?
Adapting human MRI to small animals was not an easy process. Consider, for example, the much greater power of resolution neces sary to achieve the same precision in defin ing structures in the brain of a rat as in the much larger brain of a human. Recent technological advances, however, provide SAMRI images with a resolution equal to that obtained in MRI's of the human brain and on par with the detail seen in histologi cal sections. For example, the sharpness, and thus the resolution, of an image depends in part on the geometric relationship be tween the object being imaged and the MRI chamber housing it. This chamber contains a coil that generates the magnetic field to which the subject is exposed. Special coils have been designed to perform MRI's on specific regions of the human body (e.g., the arm). These smaller coils also have been used in some animal imaging studies (Fiel and Button 1990). To obtain optimal images of organs, such as the rat brain, however, coils must be custom designed (figure 1). The coils then can be tuned to the magnetic field of a clinical scanner (Pentney et al. 1993).
Another adaptation of SAMRI was the designing of a head brace to orient and secure the rat's head within the SAMRI apparatus (Pentney et al. 1993). Precise positioning is necessary so that researchers can compare various images taken of the same rat at different times or of different rats. The threedimensional (i.e., stereotac tic) head brace incorporates three markers, which are aligned to certain anatomical parts of the rat (i.e., the teeth, ears, and top of the skull) and thus allow duplication of the crosssection (i.e., plane) of the brain being imaged. Computers convert the data into images that represent sequential 0.7 mm thick sections through the brain. The im ages of these sections then can be compiled to visualize the entire brain.

SAMRI AND THIAMINE
Thiamine, or vitamin B 1 , is essential for nor mal brain metabolism and, in turn, normal brain functioning. Several enzymes involved in the brain's metabolism of glucose-the body's primary energy source-require thiamine to function (Butterworth 1989;Butterworth et al. 1993;Langlais 1995). Thiamine deficiency has been associated with severe cognitive dysfunction, most prominently with WernickeKorsakoff syndrome (WKS), a disorder characterized by the inability to learn and remember new information for more than a few seconds (i.e., anterograde amnesia) and other cog nitive deficits (Langlais 1995).
Thiamine deficiency is relatively com mon among alcoholics, and the majority of patients diagnosed with WKS are alcohol dependent (for a review, see Langlais 1995). Alcoholism may contribute to thiamine deficiency in several ways. For example, poor nutrition, which is common in alco holics, may result in insufficient thiamine intake. Furthermore, alcohol may interfere with thiamine absorption in the intestines (Butterworth 1989). Finally, alcohol may contribute to increased thiamine excretion in the urine (Acara et al. 1983). Thus, re searchers are studying the effects of thi amine deficiency on brain structure and function to better understand alcohol's effects on the brain and the mechanisms involved in the development of WKS.
Rats often are used as a model to study the consequences of both thiamine defi ciency and alcohol abuse. To validate the rat model, however, researchers first must dupli cate observations made in human alcoholics using studies on the animals. For example, several studies with human subjects, includ ing WKS patients, have demonstrated cer tain brain changes associated with alcohol abuse (Pfefferbaum et al. 1992;Shear et al. 1994). These changes include the enlarge ment of cavities (i.e., ventricles) in the brain that produce cerebrospinal fluid. Increased ventricle size usually results from a con comitant loss of brain tissue (Pfefferbaum et al. 1992).
Pentney and colleagues (1993) inves tigated whether experimental thiamine deficiency in rats could result in similar changes in brain structure and, if so, whether SAMRI could detect these changes. Thia mine deficiency was induced in male rats by feeding them a thiaminedeficient diet for approximately 6 weeks. Control rats received a standard diet containing ade quate thiamine levels. Brain images of both groups were generated before and at select ed times during the experiment to compare the images taken from the same rat before and after the study period. Images of a lengthwise section separating the right and left sides of the brain demonstrated that after 6 weeks of thiamine deficiency, the rats' brain ventricles were significantly larger than at the beginning of the experi ment (figure 2), whereas the control animals showed no significant changes during the same period. Thiamine deficiency also resulted in extensive loss of fatty tissue and muscle in the rats' head and neck re gions. This loss, which was observed in all thiaminedeficient animals but not in the control animals, likely is attributable to inadequate glucose metabolism, which leads to lower energy production and reduced formation of fat tissue (Butter worth et al. 1993).
The increase in ventricle size induced by thiamine deficiency was even more obvious in a series of images of horizontal crosssections of a rat's brain from the top of the head to the base of the skull (figure 3). For each crosssection analyzed, the ventricles were larger after induced thi amine deficiency than before. Based on such contiguous images, computerized in tegration of the areas of interest (e.g., the ventricles) eventually will allow researchers to determine the volumes of these struc tures. Thus, SAMRI of rats confirmed earlier observations of human WKS pa tients that thiamine deficiency could in duce changes in brain structures, includ ing enlargement of the ventricles and the associated loss of brain tissue.
The SAMRI images of thiamine deficient rats also were compared with histological brain slices obtained from the same animals to determine whether SAMRI was as sensitive as histological examination in detecting morphological changes in the brain. At the end of the 6week experiment, 0.1 mmthick histological brain sections were prepared from each rat and compared with the final SAMRI image obtained from the same rat ( figure 4). This comparison showed that all the thiamine deficiency induced changes detectable in the histo logical section (e.g., the degree of increase of the ventricles) also were visible in the SAMRI image. Thus, the noninvasive SAMRI technology is a powerful and use ful substitute for histological examinations.

SAMRI AND ALCOHOL INDUCED CHANGES
As previously noted, alcohol researchers study the effects of thiamine deficiency because it frequently occurs in alcoholics and may contribute to their brain damage and neurological dysfunction (e.g., WKS). Moreover, some researchers have detected Figure 4 Comparison of a small animal magnetic resonance (SAMRI) image (left) and the histological exam ination of the corresponding brain slice (right). The SAMRI image shows the brain as well as the surrounding skull. All the brain structures that can be seen in the histological slice are vis ible in the corresponding image. increased amounts of fluid in the brains of human alcoholics (Chick et al. 1989), a change that could indicate enlarged ven tricles comparable to those observed in thiaminedeficient subjects. Based on the findings that SAMRI can detect ventricle enlargement in the brains of thiamine deficient rats, this technique is now being used to study the effects of chronic alco hol consumption on rat brain morphology.
In these studies, the rats receive a solu tion of 20percent alcohol and 80percent tap water as their only fluid source, and the control rats receive plain tap water. Over several months, the researchers take images of the animals' brains to determine if any morphological changes have occurred in the brains of the alcoholconsuming rats compared with the control rats. Prelim inary findings suggest, for example, that the ventricles are enlarged after 9 months of alcohol intake but still maintain their normal sizes after 6 months of alcohol in take. More experiments are necessary for researchers to compare adequately alcohol consuming animals with control animals and to verify that any observed differences directly result from alcohol intake and not from the animals' diet, malnutrition, or other similar factors. Nonetheless, SAMRI provides a tool for researchers to track the effects of longterm alcohol consumption under controlled conditions using an ani mal model. The findings of such studies will help elucidate the consequences of chronic alcohol consumption in humans.
New studies also are using SAMRI to investigate the effects of alcohol withdrawal and prolonged abstinence after longterm alcohol consumption. These studies will shed light on the extent to which alcohol induced changes in brain morphology are reversible. Preliminary findings indicate that enlarged ventricles observed in alcohol consuming rats may return to their original size within 6 months after the withdrawal of alcohol.
Finally, SAMRI also permits the exam ination of alcohol's effects on tissues other than the brain. For example, in preliminary studies of ventricular enlargement in the alcoholconsuming animals, the rats exhib ited wasting of muscle and fat tissue similar to that observed in the thiaminedeficient animals. Similarly, scientists will be able to use SAMRI to study alcoholinduced changes in brain structures other than the ventricles (e.g., different areas of the cortex) and the relative abundance of gray and white matter in the brain. Thus, future studies can be designed to take ample advantage of the multiplicity of information gathered through imaging of the whole animal.

CONCLUSIONS
The studies of the brains of thiamine deficient or alcoholconsuming rats de scribed above illustrate the potential of SAMRI for determining the consequences of chronic alcohol consumption and for potentially elucidating some of the causes of alcoholism. Many of these analyses cannot be conducted in humans because the experimental conditions (e.g., the amount and duration of alcohol consump tion or nutritional intake) cannot be control led adequately. Furthermore, experiments that track a disease's progress and thus take a few weeks or months to conduct in rats would require years to conduct in hu mans. The data from animals offer impor tant insights into some of the causes and consequences of alcoholism and eventually may lead to new diagnostic, prevention, and treatment approaches in humans.
In addition to providing structural in formation about various tissues by gener ating images with a clarity comparable to histological sections, MRI technology can be applied to studies of chemical and bio chemical processes and organ functions. Magnetic resonance spectroscopic imaging (MRSI) uses the same principle as MRI to determine the metabolic activity of tissues, including the brain. Combining imaging technologies such as SAMRI with analytical MRSI could offer new analytical research tools. For example, SAMRI could generate an image of a particular tissue in a living animal while MRSI simultaneously identi fies and measures biochemical compounds and processes and thus determines the rates of metabolic changes. Such analyses would reduce the time needed to conduct many experiments as well as improve the quality of the experiments conducted.
The potential applications of magnetic resonance technology are numerous and can benefit many biomedical investiga tions. For example, using animal models, researchers can track the biochemical, pathological, and physiological variables of a disease throughout the disease process. They also can evaluate the efficacy of dif ferent medications and their specific effects on each variable during the course of a dis ease. Such studies eventually will lead to the development of better medications and treatment regimens for diseases in humans.